Application of Selective TNFR1 Antagonistic Peptide Hydrostatin-SN10 in the Treatment of Sepsis

Abstract

The present invention relates to the field of biomedical technologies, and specifically to the application of a selective TNFR1 antagonist peptide Hydrostatin-SN10 derived from the venom of Hydrophis cyanocinctus in the treatment of sepsis. The present invention proposes a sepsis treatment strategy: selectively blocking the TNF-TNFR1 signaling pathway to block the cytokine storm; opening the TNF-TNFR2 signaling pathway to regulate Th17 / Treg and maintain immune balance. Hydrostatin-SN10 can significantly inhibit the expression of inflammatory factors, the phosphorylation level of key proteins in the TNFR1 pathway, and the nuclear localization of NF-κB in a macrophage inflammation model induced by LPS; effectively improve the survival rate of sepsis mice, significantly reduce the expression levels of inflammatory factors in the serum of sepsis mice, regulate immune balance, and significantly reduce the degree of inflammatory cell infiltration and tissue damage in different tissues, thereby achieving the effect of treating sepsis.

Description

Technical Field

[0001] The present invention relates to the field of biological medicine technology, and specifically, to the application of a selective TNFR1 antagonist peptide Hydrostatin-SN10 derived from the venom of Hydrophis cyanocinctus in the treatment of sepsis. Background Art

[0002] Sepsis refers to life-threatening organ dysfunction caused by a dysregulated host response to infection (Singer M, Deutschman CS, Seymour CW, et al. The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). The Journal of the American Medical Association, 2016, 315(8): 775-787.).

[0003] (1) Pathogenesis and treatment status of sepsis

[0004] The pathophysiological mechanism of sepsis is very complex. At present, it is generally believed that sepsis is caused by the imbalance between the pro-inflammatory and anti-inflammatory responses of the host infected by pathogens. In the early stage of sepsis, the main manifestation is excessive inflammatory response: pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS) stimulate immune cells to release a large number of pro-inflammatory cytokines such as TNF-α and IL-1, forming a "cytokine storm", which subsequently causes uncontrolled inflammatory tissue damage, immune dysfunction, hypermetabolic state and multiple organ dysfunction (Chousterman BG, Swirski FK, Weber GF. Cytokine storm and sepsis disease pathogenesis. Seminars in Immunopathology. 2017, 39(5): 517-28.). In the middle and late stages of sepsis, the main manifestation is excessive immunosuppression: the continuous inflammatory response consumes a large number of pro-inflammatory factors, and at the same time feedback activates the anti-inflammatory mechanism of the body, increasing the production of anti-inflammatory factors. The body gradually enters a state of immune imbalance and immunoparalysis, and lymphocyte apoptosis, functional inhibition and immune non-responsiveness occur, which severely reduces the body's resistance and immunity and aggravates the infection (Alexandra B, Annette S, Harald E, et al. Pro-Inflammatory Th1 and Th17 Cells Are Suppressed During Human Experimental Endotoxemia Whereas Anti-Inflammatory IL-10 Producing T Cells Are Unaffected. Frontiers in Immunology, 2018, 9: 1133.). The cytokine storm and immune imbalance caused by the excessive release of inflammatory mediators are considered to be the main mechanisms mediating septic organ damage and inducing MODS, thus causing patient death and poor prognosis (Pool R, Gomez H, Kellum JA. Mechanisms of organ dysfunction in sepsis. Critical Care Clinics. 2018, 34(1): 63-80.; Russell JA, Rush B, Boyd J. Pathophysiology of septic shock. Critical Care Clinics. 2018, 34(1): 43-61.).

[0005] Currently, the clinical treatment of sepsis mainly includes etiological anti-infective treatment and symptomatic supportive treatment, such as antibiotic / anti-endotoxin treatment, fluid resuscitation, the use of corticosteroids for anti-inflammation, vasoactive drugs, etc. (Gizem P, Anil UR, Elif C, et al. Sepsis and Septic Shock: Current Treatment Strategies and New Approaches. The Eurasian Journal of Medicine, 2017, 49(1): 53-58.), but it is still unable to completely improve the development and prognosis of sepsis. In particular, although glucocorticoids have anti-inflammatory and immunosuppressive effects, they still lead to secondary infections (Cronin L, Cook DJ, Carlet J, et al. Corticosteroid treatment for sepsis: a critical appraisal and meta-analysis of the literature. Critical Care Medicine. 1995, 23(8): 1430-1439.), and the mortality rate is still relatively high. There is a lack of effective therapeutic drugs for sepsis clinically.

[0006] The balance between the pro-inflammatory response and the anti-inflammatory response is crucial for the host to combat sepsis. Intervening in this delicate balance to improve the outcome of sepsis will be a major challenge. Therefore, research on treating sepsis by targeting inflammatory storms and immune imbalance has now become a hot topic. TNF-α is the most important pro-inflammatory cytokine in the early stage of inflammation, acting on two receptors, TNFR1 and TNFR2. Its pro-inflammatory and apoptotic functions are mainly mediated by TNFR1 (Brenner D, Blaser H, Mak TW. Regulation of tumour necrosis factor signalling: live or let die. Nature Reviews Immunology, 2015, 15(6): 362-374.). A related adjuvant treatment method is to remove such excessive inflammatory mediators in the body through blood purification techniques, including high-volume hemofiltration (HVHF), coupled plasma filtration adsorption (CPFA), and hemoperfusion (HP) and other techniques.In recent years, the results of multiple large-scale multicenter randomized controlled clinical trials such as IVOIRE, COMPACT, and COMPACT-2 have shown that extracorporeal blood purification therapy can effectively reduce the levels of inflammatory factors such as TNF-α in the plasma of sepsis patients. However, unfortunately, the final survival rate, organ function, and patient prognosis have not been significantly improved (Joannes-Boyau O, Honore PM, Perez P, et al. High-volume versus standard-volume haemofiltration for septic shock patients with acute kidney injury (IVOIRE study): a multicenter randomized controlled trial. Intensive Care Medicine, 2013, 39(9):1535-1546.; Livigni S, Bertolini G, Rossi C, et al. Efficacy of coupled plasma filtration adsorption (CPFA) in patients with septic shock: a multicenter randomised controlled clinical trial. BMJ Open, 2014, 4(1):e003536.; Garbero E, Livigni S, Ferrari F, et al. High dose coupled plasma filtration and adsorption in septic shock patients. Results of the COMPACT-2: a multicentre, adaptive, randomised clinical trial. Intensive Care Medicine, 2021, 47(11):1303-1311.).In addition, clinical studies have been conducted on anti-TNF-α drugs (such as monoclonal antibodies and soluble TNF receptors), but most of them have had little effect. Instead of significantly reducing the mortality rate, they may even increase it (Fisher CJ, Jr., Agosti JM, Opal SM, et al. Treatment of septic shock with the tumor necrosis factor receptor: Fc fusion protein. The Soluble TNF Receptor Sepsis Study Group. The New England Journal of Medicine, 1996, 334(26):1697-1702.; Bernard GR, Francois B, Mira JP, et al. Evaluating the efficacy and safety of two doses of the polyclonal anti-tumor necrosis factor-alpha fragment antibody AZD9773 in adult patients with severe sepsis and / or septic shock: randomized, double-blind, placebo-controlled phase IIb study. Critical Care Medicine, 2014, 42(3):504-511.). Thus, although TNF-α plays a key role in the occurrence and development of sepsis, completely blocking the biological effects of TNF-α does not bring substantial therapeutic effects. Selectively blocking the signal pathway transmitted by TNFR1 to block the harmful biological functions of TNF-α and opening the signal pathway transmitted by TNFR2 to regulate immune balance have become the research hotspots for this type of drug. Summary of the Invention

[0007] The object of the present invention is to provide an application of a selective TNFR1 antagonist peptide Hydrostatin-SN10 derived from the venom of Hydrophis cyanocinctus in the preparation of a drug for treating sepsis. The drugs used in the present invention have been disclosed in Chinese patent documents CN107090023A and CN107056921A that SN10 can treat two diseases, rheumatoid arthritis and inflammatory bowel disease, related to TNF-α. The present invention provides a new indication of Hydrostatin-SN10 - sepsis.

[0008] The overexpression of TNF-α and the abnormal activation of the TNF-TNFRs signaling pathway are closely related to T cell dysfunction and the occurrence and development of sepsis. TNFR1, also known as TNFRSF1A, CD120a or p55, has a molecular weight of 55 kDa and is widely distributed on the surface of most cells; TNFR2, also known as TNFRSF1B, CD120b or p75, has a molecular weight of 75 kDa and is only expressed in some subsets of immune cells, including regulatory T cells (Tregs) (Torrey H, Kuhtreiber WM, Okubo Y, et al. A novel TNFR2 agonist antibody expands highly potent regulatory T cells. Science Signaling, 2020, 13(661): eaba9600.).Generally speaking, TNF-TNFR1 mainly transmits pro-inflammatory and apoptotic signals; the signal network involved in TNF-TNFR2 is relatively complex, but many studies have shown that TNFR2 can activate the proliferation of Treg cells, inhibit the activation and proliferation of effector T cells, thereby playing a protective role in suppressing inflammation and maintaining the immune homeostasis of the body (Punit S, Dube PE, Liu CY, et al. Tumor Necrosis Factor Receptor 2 Restricts the Pathogenicity of CD8(+) T Cells in Mice With Colitis. Gastroenterology, 2015, 149(4): 993-1005e1002.; Yang S, Xie C, Chen Y, et al. Differential roles of TNFalpha-TNFR1 and TNFalpha-TNFR2 in the differentiation and function of CD4(+)Foxp3(+) induced Treg cells in vitro and in vivo periphery in autoimmune diseases. Cell Death and Disease, 2019, 10(1): 27.; Islam MS, Yang Y, Chen X. TNF-TNFR2 Signal Plays a Decisive Role in the Activation of CD4(+)Foxp3(+) Regulatory T Cells: Implications in the Treatment of Autoimmune Diseases and Cancer. Advances in Experimental Medicine and Biology, 2021, 1278: 257-272.).During the inflammatory storm, a large amount of released TNF-α stimulates the proliferation of Th17 cells through TNFR1 and exerts a pro-inflammatory effect, the purpose of which is to protect the body and eliminate pathogenic microorganisms. At this time, the Th17 / Treg ratio increases (Li LL, Dai B, Sun YH, Zhang TT. The activation of IL-17signaling pathwaypromotes pyroptosis in pneumonia-induced sepsis. Annals of Translational Medicine, 2020, 8(11): 674.); in the middle and late stages of sepsis, TNF-α promotes the proliferation and activation of Treg cells through TNFR2 and exerts an anti-inflammatory effect. When it exceeds the body's regulatory capacity, it causes immune imbalance, increases the Treg / Th17 ratio, and causes immunosuppression and immune paralysis (Gaborit BJ, Chaumette T, Chauveau M, et al. CirculatingRegulatory T Cells Expressing Tumor Necrosis Factor Receptor Type 2Contributeto Sepsis-Induced Immunosuppression in Patients During Septic Shock. The Journal of Infectious Diseases, 2021, 224(12):2160-2169.; Gaborit BJ, Roquilly A, Louvet C, et al. Regulatory T Cells Expressing Tumor Necrosis Factor ReceptorType 2Play a Major Role in CD4+T-Cell Impairment During Sepsis.The Journal of Infectious Diseases,2020,222(7):1222-1234.).

[0009] Based on the above research and the occurrence and development mechanism of sepsis, the present invention believes that the treatment strategy for sepsis should be: selectively block the TNF-TNFR1 signaling pathway to block the inflammatory storm; open the TNF-TNFR2 signaling pathway, regulate Th17 / Treg, and maintain immune balance.

[0010] In previous studies, a phage display library of the venom gland of Hydrophis cyanocinctus was screened using TNFR1 as a specific target, and a target-specific snake venom active peptide, Hydrostatin-SN10, was obtained. Chinese Patent Documents CN107090023A and CN107056921A disclose that Hydrostatin-SN10 can treat two diseases, rheumatoid arthritis and inflammatory bowel disease, related to TNF-α. At the same time, it is disclosed that Hydrostatin-SN10 (10AA) is target-specific and selective, binding only to TNFR1 and not to TNF-α or TNFR2; its binding ability to TNFR1 is about 2.8 μM, and it can competitively inhibit the binding of TNFR1 to TNF-α. Since SN10 can selectively and specifically bind to TNFR1, block the TNF-TNFR1 pathway, while opening the TNF-TNFR2 signaling pathway, regulate Th17 / Treg, and maintain immune balance, it is suitable for the treatment concept of sepsis. Based on this, the present invention focuses on studying the therapeutic effect of Hydrostatin-SN10 in sepsis.

[0011] The main technical solution of the present invention is: Through the RAW 264.7 cell and BMDM inflammation models induced by LPS, it is proved that Hydrostatin-SN10 has good in vitro anti-inflammatory effects; through the cecal ligation and puncture (CLP) and lipopolysaccharide (LPS)-induced sepsis animal models, it is proved that Hydrostatin-SN10 has the use for treating sepsis.

[0012] In the first aspect of the present invention, there is provided the use of the selective TNFR1 antagonist peptide Hydrostatin-SN10 in the preparation of a drug for treating sepsis.

[0013] Furthermore, the amino acid sequence of the selective TNFR1 antagonist peptide Hydrostatin-SN10 is as shown in SEQ ID NO:2; the nucleotide sequence of its encoding gene is as shown in SEQ ID NO:1.

[0014] Furthermore, for the selective TNFR1 antagonist peptide Hydrostatin-SN10, its synthesis method is: Hydrostatin-SN10 is synthesized using solid-phase peptide synthesis technology, and its purity and molecular weight are analyzed by HPLC and MS. The molecular weight is 1250.29 Daltons, and the isoelectric point is 4.39.

[0015] Furthermore, in the drug for treating sepsis, Hydrostatin-SN10 selectively antagonizes TNFR1.

[0016] Furthermore, the selective TNFR1 antagonist refers to Hydrostatin-SN10, which is specific to the target and selective. It only binds to TNFR1, does not bind to TNF-α or TNFR2, and can competitively inhibit the binding of TNFR1 to TNF-α.

[0017] Furthermore, the Hydrostatin-SN10 selectively blocks the TNF-TNFR1 signaling pathway, thereby blocking the cytokine storm in sepsis; it opens the TNF-TNFR2 signaling pathway, regulates Th17 / Treg, and maintains immune balance.

[0018] Furthermore, the drug for treating sepsis is: the selective TNFR1 antagonist peptide Hydrostatin-SN10 as the sole active ingredient, or a pharmaceutical composition containing the selective TNFR1 antagonist peptide Hydrostatin-SN10.

[0019] Furthermore, the pharmaceutical composition and conventional pharmaceutical excipients in pharmacy are made into pharmaceutical preparations.

[0020] Furthermore, the pharmaceutical preparation is a tablet, granule, dispersant, capsule, soft capsule, dripping pill, injection, powder injection or aerosol, etc.

[0021] Furthermore, the drug for treating sepsis selectively antagonizes TNFR1.

[0022] Furthermore, the drug for treating sepsis is a drug that has an inhibitory effect on the cytokine storm in sepsis and has a protective effect on multiple organs such as the liver, lung, spleen and kidney.

[0023] Furthermore, the inhibitory effect on the cytokine storm in sepsis means reducing the expression levels of organ function indexes such as AST, ALT, CRE, BUN and α-AMS in the serum of sepsis and pro-inflammatory factors such as IL-17, TNF-α, IL-6, IFN-γ, etc., and significantly increasing the expression levels of anti-inflammatory factors such as IL-10, IL-13 and IL-5.

[0024] The present invention uses LPS-induced RAW 264.7 cells and wild-type (WT), TNFR1 knockout (Tnfr1 - / - ) and TNFR2 knockout (Tnfr2 - / -)BMDM inflammation model, and observed the in vitro anti-inflammatory effect of the polypeptide provided by the present invention. The results showed that Hydrostatin-SN10 could significantly inhibit the expression of inflammatory factors such as NO, TNF-α, IL-6, and the phosphorylation of key proteins P65, IκB, JNK, ERK and P38 in the TNFR1 pathway and the nuclear localization of NF-κB in the RAW264.7 cell inflammation model stimulated by LPS, showing a good in vitro anti-inflammatory effect; Hydrostatin-SN10 could significantly inhibit the expression of WT and Tnfr2 stimulated by LPS - / - The expression of inflammatory factors such as NO, TNF-α, IL-6, and the phosphorylation of key proteins in the NF-κB and MAPK signaling pathways downstream of TNF-TNFRs in the BMDM inflammation model were significantly different from those in the control group. - / - In BMDM, Hydrostatin-SN10 had no significant alleviating effect on the above indicators, which also showed that Hydrostatin-SN10 had good anti-inflammatory effect in vitro and had specific selectivity for the TNFR1 target.

[0025] The sepsis animal model induced by CLP and LPS was used to observe the therapeutic effect of the drug provided by the present invention. The results showed that after intraperitoneal injection of Hydrostatin-SN10, Hydrostatin-SN10 could effectively improve the survival rate of septic mice; significantly reduce the expression levels of AST, ALT, CRE, BUN and α-AMS organ function indicators and pro-inflammatory factors IL-17, TNF-α, IL-6, IFN-γ, etc. in the serum of septic mice, and significantly increase the expression levels of anti-inflammatory factors IL-10, IL-13 and IL-5; HE staining analysis of liver, lung, spleen, kidney and multiple organ tissue pathology showed that Hydrostatin-SN10 can significantly reduce the degree of tissue inflammatory cell infiltration and reduce tissue damage. Overall, Hydrostatin-SN10 has an inhibitory effect on the inflammatory storm of sepsis and a protective effect on multiple organs. The detection of Foxp3 protein in spleen by immunohistochemistry showed that Hydrostatin-SN10 could significantly increase the number of Treg cells in spleen; the detection of key proteins in inflammatory signaling pathways downstream of TNFR1 by Western-blot showed that Hydrostatin-SN10 could significantly inhibit the phosphorylation of IκB, JNK, ERK and P38, thus revealing its anti-inflammatory mechanism and immunomodulatory therapeutic effect. - / - and Tnfr2 - / -C57BL / 6 mouse sepsis model was treated by intraperitoneal injection with Hydrostatin-SN10. The survival rate of the mice was observed, and it was found that Hydrostatin-SN10 could significantly improve the survival rate of CLP-induced Tnfr2- / - C57BL / 6 mice; in Tnfr1 - / - In the C57BL / 6 mouse sepsis model, Hydrostatin-SN10 did not significantly improve the survival rate of the mice, thus confirming the target selectivity of SN10 at the in vivo level.

[0026] The above research results show that the selective TNFR1 antagonist peptide Hydrostatin-SN10 has good in vitro anti-inflammatory effects and the effect of treating sepsis; at both in vitro and in vivo levels, it is clear that Hydrostatin-SN10 can selectively block the TNF-TNFR1 signaling pathway, thereby blocking the cytokine storm in the sepsis model; opening the TNF-TNFR2 signaling pathway, regulating Th17 / Treg, and maintaining immune balance.

[0027] The advantages of the present invention are as follows:

[0028] The present invention proposes a sepsis treatment strategy: selectively blocking the TNF-TNFR1 signaling pathway to block the cytokine storm; opening the TNF-TNFR2 signaling pathway to regulate Th17 / Treg and maintain immune balance. In vitro experimental studies found that Hydrostatin-SN10 could significantly inhibit the expression of inflammatory factors, the phosphorylation level of key proteins in the TNFR1 pathway, and the nuclear localization of NF-κB in the macrophage inflammation model induced by LPS; in vivo experimental studies showed that Hydrostatin-SN10 could effectively improve the survival rate of sepsis mice, significantly reduce the expression level of inflammatory factors in the serum of sepsis mice, regulate immune balance, and significantly reduce the infiltration of inflammatory cells and the degree of tissue damage in different tissues, thereby achieving the effect of treating sepsis. The present invention has found an effective drug for treating sepsis. Brief Description of the Drawings

[0029] Figure 1 Shows the effect of Hydrostatin-SN10 on the NO release of RAW 264.7 cells co-stimulated by LPS and IFN-γ. *p < 0.05, **p < 0.01.

[0030] Figure 2 Shows the effect of Hydrostatin-SN10 on the expression of inflammatory factors in the LPS-induced RAW 264.7 cell inflammation model. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0031] Figure 3Effect of Hydrostatin-SN10 on the expression of inflammatory factors in the LPS-induced RAW 264.7 cell inflammation model. *p<0.05, ***p<0.001, ****p<0.0001.

[0032] Figure 4 Effect of Hydrostatin-SN10 on the MAPK and NF-κB pathways in the LPS-induced RAW 264.7 inflammation model. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0033] Figure 5 Effect of Hydrostatin-SN10 on NF-κB nuclear localization.

[0034] Figure 6 Effect of Hydrostatin-SN10 on the release of WT, Tnfr1 - / - 、Tnfr2 - / - BMDMNO stimulated by LPS and IFN-γ. *p<0.05, **p<0.01, ***p<0.001.

[0035] Figure 7 Effect of SN10 on the expression of inflammatory factors in the LPS-induced WT BMDM inflammation model. *p<0.05, **p<0.01, ***p<0.001.

[0036] Figure 8 Effect of Hydrostatin-SN10 on LPS-induced Tnfr1 - / - Effect of Hydrostatin-SN10 on the expression of inflammatory factors in the BMDM inflammation model.

[0037] Figure 9 Effect of Hydrostatin-SN10 on LPS-induced Tnfr2 - / - Effect of Hydrostatin-SN10 on the expression of inflammatory factors in the BMDM inflammation model. *p<0.05, **p<0.01, ***p<0.001, ****p<0.001.

[0038] Figure 10 Effect of Hydrostatin-SN10 on the expression of inflammatory factors in the LPS-induced WT BMDM inflammation model. *p<0.05, **p<0.01, ***p<0.001, ****p<0.001.

[0039] Figure 11 Effect of Hydrostatin-SN10 on LPS-induced Tnfr1 - / -Effect of inflammatory factor expression in the BMDM inflammation model.

[0040] Figure 12 is the effect of Hydrostatin-SN10 on Tnfr2 induced by LPS - / - Effect of inflammatory factor expression in the BMDM inflammation model. *p<0.05, **p<0.01, ***p<0.001, ****p<0.001.

[0041] Figure 13 is the effect of Hydrostatin-SN10 on the MAPK and NF-κB pathways in the LPS-induced WT BMDM inflammation model. *p<0.05, **p<0.01, ***p<0.001.

[0042] Figure 14 is the effect of Hydrostatin-SN10 on Tnfr1 induced by LPS - / - Effect of Hydrostatin-SN10 on the MAPK and NF-κB pathways in the BMDM inflammation model.

[0043] Figure 15 is the effect of Hydrostatin-SN10 on Tnfr2 induced by LPS - / - Effect of Hydrostatin-SN10 on the MAPK and NF-κB pathways in the BMDM inflammation model. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0044] Figure 16 is the effect of Hydrostatin-SN10 on the survival rate of the CLP-induced WT C57BL / 6 mouse sepsis model. Among them, Figure A shows the modeling and administration time, and Figure B shows the survival rate of the model mice in different administration groups; *p<0.05, **p<0.01, ***p<0.001.

[0045] Figure 17 is the effect of Hydrostatin-SN10 on Tnfr1 induced by CLP - / - Effect of Hydrostatin-SN10 on the survival rate of the C57BL / 6 mouse sepsis model. Among them, Figure A shows the modeling and administration time, and Figure B shows the survival rate of the model mice in different administration groups.

[0046] Figure 18 is the effect of Hydrostatin-SN10 on Tnfr2 induced by CLP - / - Effect of Hydrostatin-SN10 on the survival rate of the C57BL / 6 mouse sepsis model. Among them, Figure A shows the modeling and administration time, and Figure B shows the survival rate of the model mice in different administration groups; *p<0.05, ***p<0.001.

[0047] Figure 19 Effect of Hydrostatin-SN10 on the absolute number of peripheral blood monocytes in a sepsis model of CLP-induced WT C57BL / 6 mice. *p < 0.05, **p < 0.01.

[0048] Figure 20 Effect of Hydrostatin-SN10 on blood biochemical indexes in a sepsis model of CLP-induced WT C57BL / 6 mice. *p < 0.05, **p < 0.01, ***p < 0.001.

[0049] Figure 21 Effect of Hydrostatin-SN10 on serum inflammatory factors in a sepsis model of CLP-induced WT C57BL / 6 mice. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0050] Figure 22 Effect of Hydrostatin-SN10 on pathological changes in lung tissues of a sepsis model of CLP-induced WT C57BL / 6 mice (×200 magnification).

[0051] Figure 23 Effect of Hydrostatin-SN10 on pathological changes in liver tissues of a sepsis model of CLP-induced WT C57BL / 6 mice (×200 magnification).

[0052] Figure 24 Effect of Hydrostatin-SN10 on pathological changes in kidney tissues of a sepsis model of CLP-induced WT C57BL / 6 mice (×200 magnification).

[0053] Figure 25 Effect of Hydrostatin-SN10 on pathological changes in spleen tissues of a sepsis model of CLP-induced WT C57BL / 6 mice (×200 magnification).

[0054] Figure 26 Effect of Hydrostatin-SN10 on the level change of spleen Treg cells in a sepsis model of CLP-induced WT C57BL / 6 mice (×200 magnification).

[0055] Figure 27 Effect of Hydrostatin-SN10 on the MAPK and NF-κB pathways in hepatocytes of a sepsis model of CLP-induced WT C57BL / 6 mice. *p < 0.05, **p < 0.01, ***p < 0.001.

[0056] Figure 28Effect of Hydrostatin-SN10 on the survival rate of LPS-induced sepsis model in WT C57BL / 6 mice. Among them, Figure A shows the modeling and administration time, and Figure B shows the survival rate of model mice in different administration groups; *p<0.05, **p<0.01.

[0057] Figure 29 Effect of Hydrostatin-SN10 on blood biochemical indexes of LPS-induced sepsis model in WT C57BL / 6 mice. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0058] Figure 30 Effect of Hydrostatin-SN10 on serum inflammatory factors of LPS-induced sepsis model in WT C57BL / 6 mice. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0059] Figure 31 Effect of Hydrostatin-SN10 on pathological changes of lung tissue in LPS-induced sepsis model of WT C57BL / 6 mice (×200 magnification).

[0060] Figure 32 Effect of Hydrostatin-SN10 on pathological changes of liver tissue in LPS-induced sepsis model of WT C57BL / 6 mice (×200 magnification).

[0061] Figure 33 Effect of Hydrostatin-SN10 on pathological changes of kidney tissue in LPS-induced sepsis model of WT C57BL / 6 mice (×200 magnification).

[0062] Figure 34 Effect of Hydrostatin-SN10 on pathological changes of spleen tissue in LPS-induced sepsis model of WT C57BL / 6 mice (×200 magnification).

[0063] Figure 35 Effect of Hydrostatin-SN10 on the change of spleen Treg cell level in LPS-induced sepsis model of WT C57BL / 6 mice (×200 magnification). Detailed implementation manners

[0064] The following further describes the detailed implementation manners provided by the present invention in conjunction with the embodiments.

[0065] The experimental methods in the following embodiments are all conventional methods unless otherwise specified.

[0066] In the following examples, Hydrostatin-SN10 used was synthesized by Qiangyao Biotechnology Co., Ltd. and its purity was ≥98% detected by HPLC; LPS powder was purchased from Sigma Company in the United States, with the model Sigma Eschericia Coli 055:B5; mouse granulocyte colony-stimulating factor (M-CSF) and mouse interferon-γ (IFN-γ) were purchased from Peprotech Company; infliximab (IFX) powder was purchased from Cilag AG Company.

[0067] Example 1: In vitro anti-inflammatory effect and mechanism of Hydrostatin-SN10 on LPS-induced RAW 264.7 cell inflammation model

[0068] The specific implementation steps are as follows:

[0069] An RAW 264.7 cell inflammation model was established by LPS induction. TNF-α antibody IFX was used as a positive control drug. Three concentration gradients of SN10, namely 25 μM, 50 μM, and 100 μM, were set for drug administration. Samples were taken at different time points after modeling according to different detection indicators to evaluate the in vitro anti-inflammatory effect of SN10 on RAW 264.7 cells and explore its anti-inflammatory mechanism.

[0070] (1) RAW 264.7 cells were co-stimulated with LPS and interferon γ, and the corresponding concentration of the drug was given to the drug administration group. After 24 h, the release amount of the inflammatory index nitric oxide (NO) was detected with Griess reagent. The results are as Figure 1 shown: Compared with the model group, SN10 could inhibit the release of NO, and the effect was similar to that of IFX.

[0071] (2) After pre-incubating with IFX and different concentrations of SN10 for 30 min, RAW 264.7 cells were stimulated with LPS to induce an inflammation model. After 6 h, the mRNA of RAW 264.7 cells was extracted, and the cell culture supernatant was collected. The expression of inflammatory factors was analyzed at the transcriptional and translational levels by RT-PCR technology and flow cytometry. The results are as Figure 2 、 3 shown: Compared with the model group, SN10 could inhibit the release of inflammatory factors such as IL-6, TNF-α, and IL-1β, and the effect was similar to that of IFX.

[0072] (3) After pre-incubating with IFX and different concentrations of SN10 for 30 min, RAW 264.7 cells were stimulated with LPS to induce an inflammation model. After 6 h, the proteins of RAW 264.7 cells were extracted, and the key proteins of the TNF-TNFRs downstream NF-κB and MAPK signaling pathways were explored by Western-blot experiment. The results are asFigure 4 As shown: Compared with the model group, SN10 can inhibit the phosphorylation of P65, IκB, JNK, ERK, and P38, and the effect is better than that of the IFX group.

[0073] (4) After pre-incubating with SN10 and IFX for 30 min, the inflammatory response of RAW 264.7 cells was induced by LPS. After 6 h, the nuclear localization of NF-κB was detected by immunofluorescence technology. The results were as Figure 5 shown: Compared with the model group, SN10 can inhibit the nuclear entry of NF-κB, and the inhibitory effect is better than that of the IFX group.

[0074] Example 2: Study on the in vitro anti-inflammatory effect, mechanism of action and target selectivity of Hydrostatin-SN10 on the LPS-induced WT, Tnfr1 - / - , Tnfr2 - / - BMDM inflammatory model

[0075] The specific implementation steps are as follows:

[0076] BMDM inflammatory models of wild-type, Tnfr1- and Tnfr2-knockout C57BL / 6 mice were established by LPS induction. TNF-α antibody IFX was used as a positive control drug, and three concentration gradients of SN10 at 25 μM, 50 μM, and 100 μM were set for drug administration. Samples were taken at different time points after modeling according to different detection indexes to evaluate the in vitro anti-inflammatory effect of SN10, verify its target selectivity and explore its anti-inflammatory mechanism.

[0077] (1) Three genotypes of BMDM were stimulated with LPS and interferon-γ (IFN-γ). Different dose administration groups were given corresponding concentrations of drugs for treatment. After 24 h, the release amount of the inflammatory index NO was detected by Griess reagent. The results were as Figure 6 shown: In WT and Tnfr2 - / - BMDM, compared with the model group, SN10 can inhibit the release of NO, and the effect is better than that of the IFX group; while in Tnfr1 - / - BMDM, neither SN10 nor IFX had an obvious alleviating effect on this index.

[0078] (2) After pre-incubating with IFX and different concentrations of SN10 for 30 min, the inflammatory models of three genotypes of BMDM were induced by LPS. After 6 h of stimulation, the mRNA of BMDM was extracted and the cell culture supernatant was collected. The inhibitory effect of SN10 on inflammatory factors in the LPS-stimulated inflammatory models of three genotypes of BMDM was analyzed by RT-PCR technology and flow cytometry at the transcriptional and translational levels, respectively. The results were as Figures 7 - 12 shown: In WT and Tnfr2 - / -In BMDMs, compared with the model group, SN10 could inhibit the release of inflammatory factors such as IL-6, TNF-α, and IL-1β, and the effect was better than that of the IFX group; while in Tnfr1 - / - In BMDMs, neither SN10 nor IFX had an obvious alleviating effect on various inflammatory factors.

[0079] (3) After pre-incubating with IFX and different concentrations of SN10 for 30 min, a BMDM inflammation model of three genotypes was induced with LPS, and the proteins of BMDMs were extracted after 6 h of stimulation. The effects of SN10 on the key proteins of the NF-κB and MAPK signaling pathways downstream of TNF-TNFRs were explored by Western-blot experiments, and the results were as follows Figures 13 - 15 shown: In WT and Tnfr2 - / - In BMDMs, compared with the model group, SN10 could inhibit the phosphorylation of P65, IκB, JNK, ERK, and P38, and the effect was better than that of the IFX group; while in Tnfr1 - / - In BMDMs, no obvious alleviating effect was detected for SN10 and IFX.

[0080] Example 3: Study on the treatment and mechanism of action of Hydrostatin-SN10 on LPS- and CLP-induced mouse sepsis models

[0081] The specific implementation steps are as follows:

[0082] 1. Construct wild-type C57BL / 6 mouse sepsis models induced by CLP (severe modeling, 75% cecal ligation) and intraperitoneal injection of LPS (severe modeling, 15 mg / kg). Different concentrations of SN10 (400 μg / kg, 800 μg / kg, and 1.6 mg / kg), IFX (4 mg / kg), and the negative control drug random peptide (RP, 800 μg / kg) were used for treatment by intraperitoneal injection 2 h before modeling, 1 h after modeling, and 2.5 h after modeling. The survival rates of mice in each group were observed, and the results were as follows Figure 16 、 28 shown: After treatment with SN10, the survival rate of mice was significantly increased compared with the model group (0%), and it showed a dose-dependent manner. The survival rates reached 50% in the 800 μg / kg group and the 1.6 mg / kg group (p < 0.01), and the effect was far better than that of the IFX group (12.5%); in the LPS-induced severe sepsis model of C57BL / 6 mice, after treatment with SN10, the survival rate of mice was also significantly increased compared with the model group (0%), and it showed a dose-dependent manner. The survival rate reached 50% in the 800 μg / kg group (p < 0.01), and the effect was far better than that of the IFX group (25%)

[0083] 2. A sepsis model was established in wild-type C57BL / 6 mice induced by CLP (moderate modeling, 50% cecal ligation) and intraperitoneal injection of LPS (moderate modeling, 10 mg / kg). The optimal drug concentrations of SN10 (800 μg / kg) and IFX (4 mg / kg) screened out by the survival rate experiment results were used for treatment by intraperitoneal injection 2 h before modeling, 1 h after modeling, and 2.5 h after modeling. The mice were sacrificed after 24 h, and the biological samples were analyzed. The results showed that:

[0084] (1) Routine blood tests were performed on the peripheral blood of CLP model mice to study the effect of the drugs on the absolute number of monocytes in the blood of mice. The results were as Figure 19 shown: Compared with the model group, the number of monocytes in the SN10 group and the IFX group was significantly reduced, indicating that SN10 could inhibit the increase in the number of monocytes (p < 0.05), and the therapeutic effect was similar to that of IFX (p > 0.05).

[0085] (2) Blood biochemical index kits were used to detect the expression of AST, ALT, CRE, BUN, and α-AMS in the sera of the two model mice to study the inhibitory effect of the drugs on the organ function damage of mice. The results were as Figure 20 、 29 shown: Compared with the model group, the contents of AST, ALT, CRE, BUN, and α-AMS in the sera of the SN10 group were significantly reduced. In the IFX group, all except the CRE index were lower than those in the model group, indicating that SN10 could reduce the damage to the liver, kidney, and pancreas, and the effect was better than that of IFX.

[0086] (3) Multifactor flow cytometry kits were used to determine the expression of inflammatory factors such as IL-6, TNF-α, and IL-1β in the sera of the two model mice, and the in vivo anti-inflammatory effects of SN10 and IFX were observed from the expression levels of inflammatory factors. The results were as Figure 21 、 30 shown: Compared with the model group, the contents of inflammatory factors such as IL-6, TNF-α, IL-1β, and IL-17 in the sera of the SN10 group were significantly reduced, and the anti-inflammatory factors such as IL-10 were significantly increased; in the IFX group, the above-mentioned inflammatory factors were reduced, but only some anti-inflammatory factors were increased and not significantly. It shows that SN10 can further affect the expression of other inflammation-related factors in the body by reducing the pro-inflammatory function of TNF-α, and the effect is better than that of IFX.

[0087] (4) HE staining was used to observe the inflammatory cell infiltration and damage in the liver, lung, kidney, and spleen tissues of the two model mice, and the therapeutic effects of SN10 and IFX were observed from the multi-organ pathological sections. The results were as Figures 22 - 25As shown in Figures 31 - 34: In the normal group of mice, the structures of various organs were intact, and no obvious inflammatory cell infiltration was seen in the tissues; in the model group, inflammatory cell infiltration was seen in various organs, the structures were severely damaged to varying degrees, and even tissue necrosis occurred; in the SN10 group and the IFX group, the inflammatory cell infiltration was significantly reduced, and the tissue damage was not severe, but the effect of SN10 was better than that of IFX.

[0088] (5) Immunohistochemistry was used to measure the expression level of FOXP3 in the spleens of two model mice to study the anti - inflammatory mechanism of SN10. The results were as Figure 26 、 35 shown: Compared with the model group, the expression level of Foxp3 in the SN10 group was significantly increased, and the increase in the IFX group was relatively not obvious. This indicates that the expression level of Treg cells in the SN10 group is higher. Combining with the results of the expression level of IL - 17 in the serum, SN10 may play an anti - inflammatory role by regulating the change of Treg / Th17, thereby inhibiting the occurrence and development of sepsis. Comparing the two results of the SN10 group and the IFX group, the SN10 group was significantly better than the IFX group, probably because compared with IFX, SN10 only blocks TNFR1 and does not affect the immune regulatory function of TNFR2.

[0089] (6) Western - blot experiments were used to detect the phosphorylation levels of key proteins in the NF - κB and MAPK signaling pathways downstream of TNF - TNFRs in liver tissues, observe the anti - inflammatory treatment effect of SN10 and reveal the in - vivo anti - inflammatory mechanism of SN10. The results were as Figure 27 shown: Compared with the model group, the SN10 group and the IFX group could significantly inhibit the phosphorylation of P65, IκB, JNK, ERK and P38, and the inhibitory effect of SN10 was better than that of the IFX group.

[0090] 3. A sepsis model of Tnfr1 - and Tnfr2 - gene - knockout C57BL / 6 mice induced by CLP (severe modeling, 75% cecal ligation degree) was constructed. Different concentrations of SN10 (400 μg / kg, 800 μg / kg and 1.6 mg / kg), IFX (4 mg / kg) and the negative control drug random peptide (RP, 800 μg / kg) were used for intraperitoneal injection at 2 h before modeling, 1 h after modeling and 2.5 h after modeling to observe the survival rate of mice in each group, so as to explore the in - vivo mechanism of action of SN10 and judge whether it has selectivity for TNFR1. The results were as Figure 17 、 18 shown: In the Tnfr2 induced by CLP - / -In the severe sepsis model of C57BL / 6 mice, after treatment with SN10, the survival rate of the mice was significantly increased compared with the model group (0%), and showed a dose-dependence. The survival rate reached 62.5% in the 800 μg / kg group (p < 0.001), and the effect was far better than that of the IFX group (25%); in Tnfr1 - / - In the sepsis model of C57BL / 6 mice, neither SN10 nor IFX had a significant effect on improving the survival rate of the mice.

[0091] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments. Those skilled in the art can also make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application. Sequence Listing <110> 8+1 Pharmaceutical Co., Ltd. <120> Application of Selective TNFR1 Antagonistic Peptide Hydrostatin-SN10 in the Treatment of Sepsis <130> <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 30 <212> DNA <213> Artificial Sequence (Artificial) <400> 1 gacgaacaac acctagagac cgaactacac 30 <210> 2 <211> 10 <212> PRT <213> Artificial Sequence (Artificial) <400> 2 Asp Glu Gln His Leu Glu Thr Glu Leu His 1 5 10

Claims

1. Use of the selective TNFR1 antagonist peptide Hydrostatin-SN10 in the preparation of a medicament for treating sepsis; the amino acid sequence of the selective TNFR1 antagonist peptide Hydrostatin-SN10 is as shown in SEQ ID NO:

2.

2. The use according to claim 1, wherein, in the medicament for treating sepsis, Hydrostatin-SN10 selectively antagonizes TNFR1.

3. The use according to claim 2, wherein, the selective antagonism of TNFR1 means that Hydrostatin-SN10 only binds to TNFR1, does not bind to TNF-α and TNFR2, and can competitively inhibit the binding of TNFR1 to TNF-α.

4. The use according to claim 1, wherein, the medicament for treating sepsis is: the selective TNFR1 antagonist peptide Hydrostatin-SN10 as the sole active ingredient, or a pharmaceutical composition containing the selective TNFR1 antagonist peptide Hydrostatin-SN10.

5. The use according to claim 4, wherein, the pharmaceutical composition and conventional pharmaceutical excipients in pharmacy are made into a pharmaceutical preparation.

6. The use according to claim 5, wherein, the pharmaceutical preparation is a tablet, granule, dispersant, capsule, dripping pill, injection, powder injection or aerosol.

7. The use according to claim 1, wherein, the medicament for treating sepsis is a medicament that has an inhibitory effect on the inflammatory storm of sepsis and has a protective effect on multiple organs such as the liver, lung, spleen and kidney.

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